We will use metalloprotein design and redesign to address important functional features common to many copper metalloenzymes. Copper proteins are an important class of metallobiomolecules with at least a dozen enzymes in the human body utilizing copper for structure and function. Most human copper enzymes are classified as oxidoreductases and are involved in an array of processes, such as electron transfer, and oxidation or reduction of substrates. Common roles of copper in these proteins are electron transfer, such as in ceruloplasmin (CP) or cytochrome c oxidase (CcO), and reactivity with O2, such as in peptidyl ?-hydroxylating monooxygenase (PHM), tyrosinase, or amine oxidases. Therefore, themes common among Cu enzymes include 1) Cu mediated electron transfer and 2) Cu catalyzed oxygen chemistry with both oxidation and reduction of substrate. The goal of this proposal is to use protein design and redesign to explore and elucidate these common themes of Cu metalloenzymes. We will accomplish this by modeling the family of noncoupled dinuclear copper proteins, which contain both an electron transfer center and a catalytic center. We therefore will model the di-copper centers of PHM, dopamine ?-monooxygenase (DBM), and nitrite reductase (NiR) using the protein azurin as the ligand or scaffold. Using the protein design methodology, we will model these enzymes structurally as well as functionally. We will examine and adjust different layers of structural elements in our models that are hypothesized to be essential for activity in the native systems. These include 1) modulating the copper redox potentials of the two Cu sites, 2) tuning the electron transfer pathway between the Cu sites, and 3) making mutations to encourage substrate binding in our models. All of these components taken together are attempts to couple together and test different factors that are deemed important for the proper function of a catalytic Cu enzyme: the design of a structurally adequate copper site, the tuning of redox potential, the modification of electron transfer paths, and the binding of substrates such as oxygen. The long term goal of the research is to understand these properties of Cu metalloenzymes and shed light on the mechanisms of the native systems. Equally as important a goal for these projects is the mentoring and training of undergraduate and M.S. students for successful careers in multidisciplinary biochemical sciences. PUBLIC HEALTH RELEVANCE: There are over a dozen human copper enzymes, many of which utilize copper to facilitate electron transfer reactions and oxidize substrate with molecular oxygen. We will use protein metal site design to incorporate and adjust different layers of structural elements into models of native systems that test factors hypothesized to be essential for function. Defining the features that lead to activity of copper in these enzymes can contribute to an understanding of factors underlying various diseases.